Reversible methylation of DNA is a powerful and ubiquitous mechanism for dynamic control of gene expression. The recent discovery that deoxy-5-methylcytosine (d5mC) in mammalian DNA can be iteratively oxidized by ten eleven translocation (TET) proteins to generate deoxy-5-hydroxymethylcytosine (d5hmC), deoxy-5-formylcytosine (d5fC), and deoxy-5-carboxycytosine (d5caC), marks a breakthrough in the field of epigenetics. These oxidized bases are predicted to be both novel epigenetic markers and part of the d5mC demethylation pathway. Mammalian TETs (mTETs) belong to the FeII/a-ketoglutarate-dependent dioxygenase (FeaKGO) family, which is widely distributed across all three kingdoms of life, including the heterolobosean amoeboflagellate, Naegleria gruberi. Little is known about the mechanistic and structural bases for substrate specificity and product profile in members of the FeaKGO TET/JBP subfamily, with oxidative activities on 5-methylpyrimidines in nucleic acids, due to challenges in in vitro protein production of these enzymes. Herein, we report the first biochemical and structural characterization of the N. gruberi protein, 5-methylpyrimidine dioxygenase 1 (mYOX1), which bears ~ 39 % similarity to mTETs. Similar to mTETs, mYOX1 iteratively oxidizes d5mC on DNA to d5caC in vitro. Our structure and detailed kinetic studies offer novel insight into the mode of substrate recognition and product release by mYOX1. Furthermore, the ease of production of mYOX1 as a recombinant protein in Escherichia coli renders it an ideal molecular diagnostic tool for the detection and mapping of oxidized d5mC epigenetic markers in various genomic contexts.

Biochemical and structural insight into the iterative oxidation mechanism of deoxy-5-methylcytosine on DNA by a Naegleria gruberi enzyme, 5-methylpyrimidine dioxygenase 1 (mYOX1)

247th National Meeting (2014)

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